Seong-Jin Yu

Novel GLP-1R/GIPR co-agonist “twincretin” is neuroprotective in cell and rodent models of mild traumatic brain injury

ABSTRACT Several single incretin receptor agonists that are approved for the treatment of type 2 diabetes mellitus (T2DM) have been shown to be neuroprotective in cell and animal models of neurodegeneration. Recently, a synthetic dual incretin receptor agonist, nicknamed “twincretin,” was shown to improve upon the metabolic benefits of single receptor agonists in mouse and monkey models of T2DM. In the current study, the neuroprotective effects of twincretin are probed in cell and mouse models of mild traumatic brain injury (mTBI), a prevalent cause of neurodegeneration in toddlers, teenagers and the elderly. Twincretin is herein shown to have activity at two different receptors, dose‐dependently increase levels of intermediates in the neurotrophic CREB pathway and enhance viability of human neuroblastoma cells exposed to toxic concentrations of glutamate and hydrogen peroxide, insults mimicking the inflammatory conditions in the brain post‐mTBI. Additionally, twincretin is shown to improve upon the neurotrophic effects of single incretin receptor agonists in these same cells. Finally, a clinically translatable dose of twincretin, when administered post‐mTBI, is shown to fully restore the visual and spatial memory deficits induced by mTBI, as evaluated in a mouse model of weight drop close head injury. These results establish twincretin as a novel neuroprotective agent and suggest that it may improve upon the effects of the single incretin receptor agonists via dual agonism. Graphical abstract Figure. No caption available. HighlightsThe dual GLP‐1/GIP receptor agonist “twincretin” was evaluated for neurotrophic and neuroprotective actions in cell and in vivo models of mild TBI.Twincretin provided neurotrophic properties in immortal human SH‐SY5Y neuronal cells ‐ elevating cAMP levels and pCREB.Twincretin provided neuroprotection against oxidative stress and glutamate excitotoxicity in human SH‐SY5Y cells.Twincretin protected rat primary cultures of dopaminergic ventral mesencephalon neurons from 6‐hydroxydopamine‐induced injury.A clinically translatable dose of twincretin fully mitigated mild TBI‐induced spatial and visual memory deficits in a mouse close head injury model.Twincretin mediated mitigation of cellular and in vivo TBI‐induced impairments highlight the agent for evaluation in neurodegenerative disorders.

Time-Dependent Protection of CB2 Receptor Agonist in Stroke

Recent studies have indicated that type 2 cannabinoid receptor (CB2R) agonists reduce neurodegeneration after brain injury through anti-inflammatory activity. The purpose of this study was to examine the time-dependent interaction of CB2R and inflammation in stroke brain. Adult male rats were subjected to right middle cerebral artery occlusion (MCAo). CB2R mRNA expression was significantly elevated >20 fold on day 2, peaked >40-fold on day 5, and normalized on day 10 post-stroke. Inflammatory markers IBA1 and TLR4 were significantly upregulated 15 fold until day 5 after MCAo. Because of the delayed upregulation of CB2R and IBA1, we next treated animals daily with CB2R agonist AM1241 or anti-inflammatory PPAR-γ agonist pioglitazone from 2 to 5 days after MCAo. Delayed treatment with pioglitazone significantly reduced abnormal neurological scores and body asymmetry as well as brain infarction in stroke animals. No behavioral improvement or reduction in brain infarction was found in animals receiving AM1241. Pioglitazone, but not AM1241, significantly reduced IBA1 expression in the stroke cortex, suggesting that delayed treatment with AM1241 failed to alter ischemia-mediated IBA-1 upregulation. In contrast, pretreatment with AM1241 significantly reduced brain infarction and neurological deficits. In conclusion, our data support a time-dependent neuroprotection of CB2 agonist in an animal model of stroke. Delayed post- treatment with PPAR-γ agonist induced behavioral recovery and microglial suppression; early treatment with CB2R agonist suppressed neurodegeneration in stroke animals.

Transplantation of Human Placenta-Derived Multipotent Stem Cells Reduces Ischemic Brain Injury in Adult Rats

After the onset of stroke, a series of progressive and degenerative reactions, including inflammation, is activated, which leads to cell death. We recently reported that human placenta-derived multipotent stem cells (hPDMCs) process potent anti-inflammatory effects. In this study, we examined the protective effect of hPDMC transplants in a rodent model of stroke. Adult male Sprague–Dawley rats were anesthetized. hPDMCs labeled with a vital dye of fluorescing microparticles, DiI, or vehicle were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Five minutes after grafting, the right MCA was transiently occluded for 60 min. Stroke animals receiving hPDMCs showed a significant behavioral improvement and reduction in lesion volume examined by T2-weighted images 4 days poststroke. Brain tissues were collected 1 day later. Human-specific marker HuNu immunoreactivity and DiI fluorescence were found at the hPDMC graft sites, suggesting the survival of hPDMCs in host brain. Grafting of hPDMCs suppressed IBA1 immunoreactivity and deramification of IBA1+ cells in the perilesioned area, suggesting activation of microglia was attenuated by the transplants. Taken together, our data indicate that hPDMC transplantation reduced cortical lesions and behavioral deficits in adult stroke rats, and these cells could serve as a unique anti-inflammatory reservoir for the treatment of ischemic brain injury.

A novel CXCR4 antagonist CX549 induces neuroprotection in stroke brain

C-X-C chemokine receptor type 4 (CXCR4) is a receptor for a pleiotropic chemokine CXCL12. Previous studies have shown that the acute administration of the CXCR4 antagonist AMD3100 reduced neuroinflammation in stroke brain and mobilized bone marrow hematopoietic stem cells (HSCs). The purpose of this study was to characterize the neuroprotective and neurotrophic effect of a novel CXCR4 antagonist CX549. We demonstrated that CX549 had a higher affinity for CXCR4 and was more potent than AMD3100 to inhibit CXCL12-mediated chemotaxis in culture. CX549 effectively reduced the activation of microglia and improved neuronal survival after injury in neuron/microglia cocultures. Early poststroke treatment with CX549 significantly improved behavioral function, reduced brain infarction, and suppressed the expression of inflammatory markers. Compared to AMD3100, CX549 has a higher affinity for CXCR4, is more efficient to mobilize HSCs for transplantation, and induces behavioral improvement. Our data support that CX549 is a potent anti-inflammatory agent, is neuroprotective against ischemic brain injury, and may have clinical implications for the treatment of stroke.

Methamphetamine induces a rapid increase of intracellular Ca++ levels in neurons overexpressing GCaMP5

In this study, methamphetamine (Meth)‐ and glutamate (Glu)‐mediated intracellular Ca++ (Ca++i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca++ probe, GCaMP5, by adeno‐associated viral (AAV) serotype 1. Binding of Ca++ to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca++i, which was blocked by MK801, suggesting that Meth enhanced Ca++i through Glu receptor in neurons. The Meth‐mediated Ca++ signal was also blocked by Mg++, low Ca++ or the L‐type Ca++ channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca++ influx but partially reduced the peak of Ca++i. These data suggest that Meth enhanced Ca++ influx through membrane Ca++ channels, which then triggered the release of Ca++ from the endoplasmic reticulum in the cytosol. AAV‐GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth‐induced green fluorescence was found in the NeuN‐containing cells in the cortex, suggesting that Meth increased Ca++ in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca++i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca++i, modulation of Ca++i may be useful to reduce Meth‐related reactions.

Neurotrophic and neuroprotective effects of oxyntomodulin in neuronal cells and a rat model of stroke.

ABSTRACT Proglucagon‐derived peptides, especially glucagon‐like peptide‐1 (GLP‐1) and its long‐acting mimetics, have exhibited neuroprotective effects in animal models of stroke. Several of these peptides are in clinical trials for stroke. Oxyntomodulin (OXM) is a proglucagon‐derived peptide that co‐activates the GLP‐1 receptor (GLP‐1R) and the glucagon receptor (GCGR). The neuroprotective action of OXM, however, has not been thoroughly investigated. In this study, the neuroprotective effect of OXM was first examined in human neuroblastoma (SH‐SY5Y) cells and rat primary cortical neurons. GLP‐1R and GCGR antagonists, and inhibitors of various signaling pathways were used in cell culture to characterize the mechanisms of action of OXM. To evaluate translation in vivo, OXM‐mediated neuroprotection was assessed in a 60‐min, transient middle cerebral artery occlusion (MCAo) rat model of stroke. We found that OXM dose‐ and time‐dependently increased cell viability and protected cells from glutamate toxicity and oxidative stress. These neuroprotective actions of OXM were mainly mediated through the GLP‐1R. OXM induced intracellular cAMP production and activated cAMP‐response element‐binding protein (CREB). Furthermore, inhibition of the PKA and MAPK pathways, but not inhibition of the PI3K pathway, significantly attenuated the OXM neuroprotective actions. Intracerebroventricular administration of OXM significantly reduced cerebral infarct size and improved locomotor activities in MCAo stroke rats. Therefore, we conclude that OXM is neuroprotective against ischemic brain injury. The mechanisms of action involve induction of intracellular cAMP, activation of PKA and MAPK pathways and phosphorylation of CREB. Graphical abstract Figure. No caption available. HighlightsWe evaluated the neurotrophic and neuroprotective actions of OXM in neuronal cultures and in an ischemic stroke model.OXM provided neurotrophic properties in immortal human SH‐SY5Y neuronal cells ‐ elevating cAMP levels and pCREB.OXM provided neuroprotection against glutamate excitotoxicity and oxidative stress injury in SH‐SY5Y cells and rat primary neurons.OXM actions were primarily mediated by the GLP‐1R but not GCGR, and involved the PKA and MAPK but not PI3K pathway.OXM significantly reduced cerebral infarct size and improved locomotor activities in a transient MCAo rat model of stroke.

Methamphetamine alters reference gene expression in nigra and striatum of adult rat brain

The nigrostriatal dopaminergic system is a major lesion target for methamphetamine (MA), one of the most addictive and neurotoxic drugs of abuse. High doses of MA alter the expression of a large number of genes. Reference genes (RGs) are considered relatively stable and are often used as standards for quantitative real-time PCR (qRT-PCR) reactions. The purpose of this study was to determine whether MA altered the expression of RGs and to identify the appropriate RGs for gene expression studies in animals receiving MA. Adult male Sprague-Dawley rats were treated with high doses of MA or saline. Striatum and substantia nigra were harvested at 2h or 24h after MA administration. The expression and stability of 10 commonly used RGs were examined using qRT-PCR and then evaluated by geNorm and Normfinder. We found that MA altered the expression of selected RGs. These candidate RGs presented differential stability in the striatum and in substantia nigra at both 2h and 24h after MA injection. Selection of an unstable RG as a standard altered the significance of tyrosine hydroxylase (TH) mRNA expression after MA administration. In conclusion, our data show that MA site- and time-dependently altered the expression of RGs in nigrostriatal dopaminergic system. These temporal and spatial factors should be considered when selecting appropriate RGs for interpreting the expression of target genes in animals receiving MA.

Post-treatment with PT302, a long-acting Exendin-4 sustained release formulation, reduces dopaminergic neurodegeneration in a 6-Hydroxydopamine rat model of Parkinson’s disease

We previously demonstrated that pretreatment with Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) –mediated dopaminergic neurodegeneration. The use of GLP-1 or Exendin-4 for Parkinson’s disease (PD) patients is limited by their short half-lives. The purpose of this study was to evaluate a new extended release Exendin-4 formulation, PT302, in a rat model of PD. Subcutaneous administration of PT302 resulted in sustained elevations of Exendin-4 in plasma for >20 days in adult rats. To define an efficacious dose within this range, rats were administered PT302 once every 2 weeks either before or following the unilaterally 6-hydroxydopamine lesioning. Pre- and post-treatment with PT302 significantly reduced methamphetamine–induced rotation after lesioning. For animals given PT302 post lesion, blood and brain samples were collected on day 47 for measurements of plasma Exendin-4 levels and brain tyrosine hydroxylase immunoreactivity (TH-IR). PT302 significantly increased TH-IR in the lesioned substantia nigra and striatum. There was a significant correlation between plasma Exendin-4 levels and TH-IR in the substantia nigra and striatum on the lesioned side. Our data suggest that post-treatment with PT302 provides long-lasting Exendin-4 release and reduces neurodegeneration of nigrostriatal dopaminergic neurons in a 6-hydroxydopamine rat model of PD at a clinically relevant dose.

Neuroprotective Action of Human Wharton’s Jelly-Derived Mesenchymal Stromal Cell Transplants in a Rodent Model of Stroke

Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) have distinct immunomodulatory and protective effects against kidney, liver, or heart injury. Limited studies have shown that WJ-MSCs attenuates oxygen–glucose deprivation-mediated inflammation in hippocampal slices. The neuroprotective effect of intracerebral WJ-MSC transplantation against stroke has not been well characterized. The purpose of this study was to examine the neuroprotective effect of human WJ-MSC (hWJ-MSC) transplants in an animal model of stroke. Adult male Sprague–Dawley rats were anesthetized and placed in a stereotaxic frame. hWJ-MSCs, pre-labeled with chloromethyl benzamide 1,1’-dioctadecyl-3,3,3’3’- tetramethylindocarbocyanine perchlorate (CM-Dil), were transplanted to the right cerebral cortex at 10 min before a transient (60 min) right middle cerebral artery occlusion (MCAo). Transplantation of hWJ-MSCs significantly reduced neurological deficits at 3 and 5 days after MCAo. hWJ-MSC transplants also significantly reduced brain infarction and microglia activation in the penumbra. Grafted cells carrying CM-Dil fluorescence were identified at the grafted site in the ischemic core; these cells were mostly incorporated into ionized calcium-binding adaptor molecule (+) cells, suggesting these xenograft cells were immuno-rejected by the host. In another set of animals, hWJ-MSCs were transplanted in cyclosporine (CsA)-treated rats. hWJ-MSC transplants significantly reduced brain infarction, improved neurological function, and reduced neuroinflammation. Less phagocytosis of CM-dil-labeled grafted cells was found in the host brain after CsA treatment. Transplantation of hWJ-MSC significantly increased glia cell line-derived neurotrophic factor expression in the host brain. Taken together, our data support that intracerebral transplantation of hWJ-MSCs reduced neurodegeneration and inflammation in the stroke brain. The protective effect did not depend on the survival of grafted cells but may be indirectly mediated through the production of protective trophic factors from the transplants.

Improving Neurorepair in Stroke Brain Through Endogenous Neurogenesis-Enhancing Drugs:

Stroke induces not only cell death but also neurorepair. De novo neurogenesis has been found in the subventricular zone of the adult mammalian brain days after stroke. Most of these newly generated cells die shortly after the insult. Recent studies have shown that pharmacological manipulation can improve the survival of endogenous neuroprogenitor cells and neural regeneration in stroke rats. As these drugs target the endogenous reparative processes that occur days after stroke, they may provide a prolonged window for stroke therapy. Here, we discuss endogenous neurogenesis-enhancing drugs and review the general status of stroke therapeutics in evaluating the field of pharmacotherapy for stroke.